The central neural mechanisms that maintain breathing and achieve blood gas homeostasis during sleep have considerable clinical relevance for congenital central hypoventilation syndrome (CCHS), central and obstructive sleep apnea (OSA), sudden infant death syndrome (SIDS) and the treatment of pain with opiates. Under the auspices of this grant, we have identified and extensively characterized a population of lower brainstem neurons (ccRTN neurons) that could be playing a pivotal role in the CO2-mediatedregulation of breathing. The activity of these neurons is exquisitely sensitive to CO2 in vivo due to the fact that they respond directly to changes in the surrounding pH and receive powerful input from the oxygen and CO2- sensing carotid bodies. Furthermore, we have shown that the ccRTN neurons provide a strong excitatory drive to the respiratory pattern generator (RPG) and that their absence eliminates RPG activity under anesthesia regardless of the level of CO2. Recent findings of major significance to CCHS suggest that the ccRTN neurons may be even more important for CO2 homeostasis in conscious mammals than anticipated. Without input from these neurons, breathing may not be possible during sleep. In 2006, we demonstrated that, in rodents, the ccRTN neurons express Phox2b, the defining mutated gene in CCHS identified in 2003. The cardinal signs of CCHS are day time hypoventilation, total sleep apnea and loss of breathing stimulation by CO2. In 2008/9, geneticists found that the ccRTN neurons are selectively absent at birth in a mouse model of CCHS. In other words, the input-output properties of the ccRTN neurons, as we understand them currently, seem precisely appropriate for these cells to contribute to CO2 homeostasis via breathing. The genetic disease, CCHS, suggests that these neurons are critical in this regard, especially during sleep. These possibilities are fascinating and deserve to be thoroughly tested, not merely for their importance in CCHS but for their control of breathing during sleep in normal animals. Accordingly, the main objectives of this renewal application are: a) to assess the importance of the ccRTN neurons for involuntary breathing during various states of vigilance, b) to determine how the ccRTN neurons control breathing, specifically which respiratory neurons they target, and c) to further examine how the activity of the ccRTN neurons is regulated with special focus on their control by serotonin, another major neurotransmitter involved in breathing. The results from the proposed experiments will help to determine whether selective stimulation of these neurons could offer a therapeutic opportunity when hypoventilation is detrimental to health, e.g. OSA. To address these issues we will continue using the large repertoire of neurophysiological and neuroanatomical methods that we have developed in prior years and we will make heavy use of a new lentiviral vector approach to manipulate ccRTN neurons selectively in vivo, a method that has allowed us to unleash the power of optogenetics for the study of cardiorespiratory control.